This invention relates to a device for multi-photon fluorescence microscopy for obtaining information from biological tissue and to a method for multi-photon fluorescence microscopy.
Such device includes a laser unit for generating an excitation radiation, an optical unit which is formed to shape the excitation radiation for generating an optical signal and focus the same at different locations in or on an object to be examined, a detector module for detecting the optical signal from the region of the object, and a signal processing and control module for the signal-technological and algorithmic processing of said optical signal for converting the same into a diagnostically evaluable image signal and for controlling the entire system.
In multi-photon fluorescence microscopy (short: multi-photon microscopy) so-called multi-photon microscopes are used, which are special optical microscopes from the group including laser scanning microscopes. High-resolution microscopic images are generated by utilizing the so-called multi-photon fluorescence (mostly two-photon fluorescence) or the generation of higher harmonics, for example frequency doubling or tripling and as a result the generation of the second or third harmonic (SHG: second harmonic generation; THG: third harmonic generation) of the incident excitation light.
In multi-photon microscopy, a strong focused excitation radiation, mostly generated by a laser, is used to generate non-linear optical effects in a tissue to be examined, which effects are based on the interaction of a plurality of photons (light particles) arriving in a molecule at the same time. The strength of the generated signal does not increase linearly with the number of photons incident per unit time, but with the square (in the case of two-photon effects) or the third power (in the case of three-photon effects). With respect to the entry of the excitation radiation into the tissue, the operation of a multi-photon microscope is similar to that of a confocal laser scanning microscope. In the confocal microscope, other than in the multi-photon microscope, remitted primary radiation and not secondary radiation is used for image formation. In the signal detection channel, the former device, other than the latter, furthermore includes a pin-hole (narrow diaphragm for eliminating remitted radiation from outside the laser focus). While because of the aforementioned particularities confocal laser scanning microscopes have a penetration depth of 50-80 μm depending on the preparation, deeper regions, e.g. down to 200 μm, in very favorable cases even down to 1000 μm, can be represented with the multi-photon microscopy, so that more meaningful pictures of living tissue, for example of skin layers of a human being, can be made.
The most widely used method for multi-photon microscopy is the two-photon fluorescence microscopy (short: two-photon microscopy). While in the conventional (single-photon) fluorescence microscopy an electron is excited in a fluorescent molecule by absorption of one photon each, i.e. is raised to a higher energy state, the excitation of the electron in the two-photon fluorescence microscopy is caused by the simultaneous or almost simultaneous absorption of two photons (two-photon absorption).
In three-photon microscopy, the excitation correspondingly is effected by three photons arriving simultaneously or almost simultaneously.
Fluorescence is obtained when dyes absorb incident (exciting) photons and subsequently again release another photon. By means of the exciting photons, an electron is raised to a higher energy level and the photon energy hence is stored temporarily. In normal fluorescence microscopy this excitation is accomplished by exactly one photon. The electron remains at the higher energy level for a few hundred picoseconds up to several nanoseconds, before it falls back again and thereby emits a new, longer-wavelength, lower-energy photon. When excitation is effected with blue light, for example, green fluorescence is obtained, as is the case with fluorescein. In two-photon microscopy, the excitation of an electron is effected by exactly two photons, which all in all have the same energy as the one excitation photon of normal fluorescence microscopy. A prerequisite for the excitation, however, consists in that the two photons arrive at the same time—within one attosecond (10−18 s)—, since there is no stable intermediate energy level of the electron to be excited.
In normal fluorescence microscopy, the exciting photon has a shorter wavelength and hence more energy than the emitted photon. In the case of multi-photon excitation, on the other hand, excitation is effected with photons which have a distinctly greater wavelength and thus less energy per photon than the emitted photons. In this way, for example, dark-red or infrared light can be used for excitation, in order to generate green fluorescence. This is possible because two or more exciting photons lead to the generation of only one emitted photon. In the two-photon excitation, the excitation wavelength approximately is about twice the normally used excitation wavelength, in the three-photon excitation three times, etc.
The fundamental concept of two-photon fluorescence microscopy is described in the publication W. Denk, J. H. Strickler, W. W. Webb “Two-Photon Laser Scanning Fluorescence Microscopy”, Science, Vol. 248, pp. 73-76 (6 Apr. 1990).
From U.S. Pat. No. 5,034,613 a device for multi-photon fluorescence microscopy is known, in which an excitation radiation generated by a laser is directed onto an object to be examined by means of movable mirrors present in the beam path. To achieve an excitation at different locations of the object and in this way form an image excited pixel by pixel, the excitation beam is changed in its position by tilting the movable mirrors such that the focus point of the excitation radiation moves through the object, excites the same location by location and thereby generates signals in the object location by location. The resulting secondary radiation (consisting of a fluorescence radiation and a possibly generated higher harmonic of the excitation radiation) is collected and detected, in order to form a complete image in one or more planes of the object with reference to the signals from the individual locations.
With the device known from U.S. Pat. No. 5,034,613 only sectional images substantially can be formed from a small segment of the object to be examined. This is due to the fact that in two-photon microscopy large apertures of the used optics are necessary because of the necessary high intensities for excitation, which require a focusing of the excitation radiation to a focus diameter of 0.5 μm up to maximally about 3 μm (this results from the laws of beam product maintenance, when optically representing laser beams). These apertures have a numerical aperture of NA=0.4 to NA=1 (or when using an immersion fluid up to NA=1.45), which corresponds to a full cone angle of the focused beam of about 50° to 135°. Such large-aperture beam bundles only can be focused by high-magnification microscope objectives or comparable complex optical systems, which inevitably only have comparatively small fields of view with a diameter of about 0.5 mm to 1 mm (field of view here is understood to be the maximum field which can be swept by a deflected excitation beam). In other words, this means: With the large apertures for focusing the excitation radiation onto the object, which are necessary for two-photon microscopy, the surface over which the excitation radiation can sweep by beam deflection necessarily is limited. By beam deflection with the use of rotary or tiltable mirrors, only fields with a diameter of maximally 1 mm can be excited, so that the recordable images are limited to edge lengths of not more than 1 mm.
To use the two-photon microscopy for example for examining a skin layer for pathological changes, such images often are too small.
Conventionally, in the two-photon microscopy a lateral scan of a plane (skin layer) initially is made and subsequently the focus depth for recording further, e.g. deeper skin layers is newly adjusted. In this way, a sequence of superimposed layers successively is recorded through the skin. From a users point of view in the case of the medical application, it would be desirable, however, to have vertical sectional images through the skin, which correspond to the cut position commonly used in histopathology, which are familiar to the medical examiners and which correspond to their diagnostic point of view.
In general, images from the skin accordingly would be desirable in a vertical cut position, which for example can fully represent a lesion extending over a length of several mm or even cm.
From EP 1 929 939 A2 an endoscopically usable device for multi-photon microscopy is known, in which the tip of an optical fiber serving as light guide with miniaturized focusing optics arranged thereon is movable in an endoscope, in order to direct an excitation radiation generated by a laser onto an object. Due to the fact that at the tip of the light guide merely an optical system of small dimension and hence also of small aperture can be used, the achievable local resolution is limited, since only comparatively large focus diameters can be achieved for local excitation. In addition, the arrangement of EP 1 929 939 A2 uses the same light guide for forwarding the excitation radiation and for returning the optical signals picked up from the object, which on the one hand places high demands on the light guide (transmission of ultra-short pulse laser radiation of high beam quality, i.e. in the TEM00 mode) and on the other hand is disadvantageous for the transmission quality and yield of the received signals. The transmitted excitation radiation additionally is deteriorated in its quality by the fiber dispersion, e.g. by enlarging the pulse duration or by the so-called “chirping”. In addition, when moving the focusing optics for recording a complete image, the light guide always must be moved as well, which renders the spatial movement quite complex and at the same time limits the same, since sectional images of great lateral expansion cannot be recorded.